Methods and products for expression of micro RNAs

ABSTRACT

The invention relates to microRNAs, methods of producing microRNAs and methods for using microRNAs.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Applicationfiled Aug. 7, 2003, entitled “METHODS AND PRODUCTS FOR EXPRESSION OFMICRO RNAs”, having Ser. No. 60/493,239 the contents of which areincorporated by reference herein in their entirety.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by grant numberGM67031 from National Institutes of Health. The Government may havecertain rights in the invention.

BACKGROUND OF THE INVENTION

MicroRNAs (miRNAs) are endogenous RNAs, some of which are known toregulate the expression of protein-coding genes at theposttranscriptional level. During miRNA maturation in animals, theprimary transcript is first processed to a stem-loop precursor and thenthe stem-loop is processed to yield a mature miRNA of about22-nucleotides. These molecules can direct the cleavage of mRNA or theycan interfere with productive translation of the mRNA, either of whichresults in reduced protein accumulation and hence the miRNAs are able tomodulate gene expression and related cellular activities. miRNAs areimportant in development and differentiation, and thus the alteredexpression of miRNAs could be used to alter development anddifferentiation during tissue engineering and other applications.Furthermore, miRNA-like stem-loops can be expressed in cells as avehicle to deliver artificial miRNAs and short interfering RNAs (siRNAs)for the purpose of modulating the expression of endogenous genes throughthe miRNA and or RNAi pathways. This can be a useful tool for studyinggene function, human therapies, and other applications. However, currentmethods for expressing miRNAs, artificial miRNAs, and siRNAs areinefficient and are not effective for many small RNA sequences.

SUMMARY OF THE INVENTION

The present invention relates in part to products and methods of makingand using microRNA molecules. In one aspect of the invention a precursormicroRNA molecule is provided. The precursor microRNA molecule is anisolated nucleic acid including a stem-loop structure wherein a microRNAsequence is incorporated into the stem-loop structure. The precursormicroRNA molecule includes a microRNA flanking sequence on either orboth sides of the microRNA sequence.

In an embodiment of the invention the microRNA sequence and the microRNAflanking sequence are derived from the same microRNA gene. In anotherembodiment of the invention the microRNA sequence and the microRNAflanking sequence are not derived from the same microRNA gene.

In another aspect the invention is a precursor microRNA molecule havinga nucleic acid having a stem-loop structure, wherein a microRNA sequenceis incorporated into a stem of the stem-loop structure, and, a microRNAflanking sequence flanking at least one end of the stem-loop structure,wherein the microRNA sequence and the microRNA flanking sequence are notderived from the same microRNA gene. Optionally the microRNA sequence isan artificial microRNA.

In one embodiment the precursor microRNA molecule includes at least twostem-loop structures. In another embodiment the microRNA sequences areat least 16-28 nucleotides in length. In another embodiment the microRNAflanking sequences are 40-4,000 or 40-2,000 nucleotides in length. Inyet another embodiment the microRNA flanking sequences are at least 40nucleotides in length.

In yet another embodiment the precursor microRNA molecule has thefollowing nucleic acid sequence

-   -   wherein X₁, and X₂ are nucleotides and wherein N₁ and N₂ are        nucleic acids of 16-28 nucleotides in length and N₁ and N₂ have        at least partial complementarity. X₁ and X₂ may each be at least        40 nucleotides.

The precursor microRNA molecule in some embodiments has microRNAflanking sequences flanking each end of the stem-loop structure.

Another aspect of the invention provides a method of altering theproductive utilization of a target mRNA. The method includes contactinga cell with a vector capable of expressing a precursor microRNA whereinthe microRNA sequence is capable of altering the productive utilizationof the target mRNA, either by specifying the cleavage of the target mRNAor by altering the accumulation of the protein of the target mRNAthrough another mechanism, such as translation repression. In oneembodiment the precursor microRNA is specific for a cancer-associatedRNA. In another embodiment the precursor microRNA is specific for aviral RNA.

In one embodiment the method of contacting a cell with a vector capableof expressing a precursor microRNA wherein the microRNA sequence iscapable of altering the productive utilization of a target mRNA isperformed in vivo. In another embodiment the method is performed in asubject having cancer. In yet another embodiment the method is performedin a subject having an infection.

In another aspect of the invention a method of altering the productiveutilization of a target mRNA including contacting a cell with a vectorcapable of expressing a mature microRNA that is not naturally expressedin the cell is provided. The mature microRNA is expressed at a levelsufficient to cause at least a 2-fold reduction in the accumulation of aprotein from the target mRNA of the target protein. In other embodimentsit is at least a 5-fold, 10-fold, 20-fold, or 30 fold reduction.

A method of altering productive utilization of a target mRNA in primarycells is also provided. The method involves contacting a primary cellwith a vector capable of expressing a mature microRNA that is notnaturally expressed in the cell, wherein the mature microRNA isexpressed at a level sufficient to cause a reduction in accumulation ofa protein from the target mRNA in the primary cell. In one embodimentthe primary cell is in vivo.

A method for modulating hematopoiesis is also provided. The methodinvolves contacting a hematopoietic cell with a vector capable ofexpressing a precursor microRNA, wherein the precursor microRNA includesa microRNA sequence capable of altering accumulation of a proteininvolved in hematopoiesis.

In yet another aspect of the invention a composition including a vectorfor producing a precursor microRNA is provided. The vector includes asequence encoding a precursor microRNA, including microRNA flankingsequences and at least one promoter element. In one embodiment thepromoter is an inducible or tissue specific promoter.

In one embodiment the vector is a viral vector. In a second embodimentthe vector is a retroviral vector. In another embodiment the vectorincludes the nucleic acid sequence of SEQ ID NO. 1 or variants thereof.

One aspect of the invention includes a host cell transfected with avector capable of producing a precursor microRNA.

Another aspect of the invention encompasses a method for detectingprecursor microRNA expression. The precursor microRNA is incorporatedinto a reporter system. This composition is transfected into a hostcell. The expression of a reporter gene product is detected to detectthe expression of precursor microRNA by its effect on the accumulationof the protein corresponding to the target mRNA. In one embodiment thereporter system includes a firefly luciferase reporter gene.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention may be more easily and completely understood whentaken in conjunction with the accompanying figures.

FIG. 1. (a) Retroviral vectors for efficient expression of microRNAhairpins. (b) Northern analysis of the expression of a ˜70-nucleotidemiR-132s hairpin from the single-copy (SC-miR-132s) or double-copy(DC-miR-132s) constructs. (c) Northern analysis of the expression ofexact microRNA hairpin using single copy and double-copy retroviralconstructs.

FIG. 2. (a) Schematic of miR-223 genes containing the miR-223 minimalhairpin and increasing amounts of flanking genomic sequences.miR-223_(—)67 is the predicted 67-nucleotide hairpin that containsmiR-223. miR-223_(—)67Si is a perfect complement hairpin derived fromthe original bulged miR-223_(—)67 hairpin. (b to d) Northern analysis ofthe expression of miR-223 genes with different length of flankingsequences in 293T cells. Total cellular RNA (b), or cytoplasmic RNA (c),or nuclear RNA (d) from 293T cells transfected with indicated miR-223expression constructs was analyzed.

FIG. 3. Examples of miRNA expression using longer miRNA genes containinga miRNA hairpin and its correspondent flanking sequences. The longermiRNA gene is about 270 nt in length, and consists of 20 nt miRNAsequence and 125 nt flanking sequence on both sides of the miRNA.Northern analysis of miR-223 (a), miR-181 (b), or miR-132s (c)expression from longer miRNA genes of ˜270 nt in length in 293T orprimary bone marrow cells.

FIG. 4. Northern analysis of the expression and maturation of miR-30from a longer precursor (272 nt) and a minimal hairpin precursor (71nt). Northern analysis of miR-30 expression and maturation using probesagainst miR-30 (a), or miR-30* (b). miR-30* is the small RNA processedfrom the opposite arm of the miR-30 hairpin precursor.

FIG. 5. A luciferase reporter system for testing microRNA-mediatedrepression in mammalian cells. (a) A schematic diagram of retroviralreporter constructs. (b) A graph to show repression of the targetreporter gene by ectopically expressed miR-223 in mammalian cells.

FIG. 6. (a) Schematic diagram of the predicted stem-loop structure ofthe B1_C02-3 miRNA. Green letters indicate the endogenous miRNAsequence. (b) Northern analysis of B1_C02-3 expression and maturationusing probes against the miRNA or the miRNA*.

FIG. 7. Schematic diagram of artificial-microRNA/siRNA design.

FIG. 8 Expression of artificial-microRNA/siRNAs using a microRNAtemplate. (a) Northern analysis of ectopically expressed SiLuc-1276 andSiLuc-1276*. (b) Northern analysis of ectopically expressed SiLuc-311and SiLuc-311*.

FIG. 9 Repression of reporter gene expression by ectopically expressedartificial-microRNA/SiRNAs.

FIG. 10. Inducible expression of B1_C02-3 microRNA at variousconcentrations of Doxycycline.

FIG. 11. Tissue and developmental expression of microRNAs cloned frommouse bone marrow. Northern analyses were used to determine microRNAexpression in different mouse tissues, including brain, heart, lung,liver, kidney, muscle, fetal liver, bone marrow, spleen, and thymus.

FIG. 12. Northern blot showing microRNA expression during hematopoieticlineage commitment.

FIG. 13. (a) Graph to show percentage Thy1.2 positive (Thy1.2⁺) cellsand CD-19 positive (CD-19⁺) cells among the differentiatinghematopoietic progenitor cells ectopically expressing no microRNA(vector), a non-hematopoietic microRNA (miR-30), or either of threehematopoietic microRNAs (miR-223, miR-132, or miR-181). (b)Representative FACS analyses of Thy-1.2 (Thy-1.2 APC) and CD-19 (CD-19PE) lineage marker expression. (c) Graph to show percentage Mac-1 andGr-1 negative cells (Mac-1⁻ Gr-1⁻), Mac-1 positive and Gr-1 negative tointermediate (Mac-1⁺ Gr-1^(−/low)), Mac-1 and Gr-1 positive (Mac-1⁺Gr-1⁺) cells among the differentiating hematopoietic progenitor cellsectopically expressing no microRNA (vector), miR-223, miR-30, miR-132,or miR-181. The average of 12 culture replicates for each construct isshown, with error bars indicating the standard deviation. (d)Representative FACS analyses of Mac-1 (Mac-1 APC) and Gr-1 (Gr-1 PE)lineage marker expression.

DETAILED DESCRIPTION

MicroRNAs (which are defined in more detail later as including siRNAsand artificial microRNAs as well as endogenous microRNAs) have potentialfor use as therapeutics as well as research tools, e.g. analyzing genefunction. Although these molecules have potential, one limitationassociated with these molecules is the difficulty in expressing adequatequantities of functional mature microRNA. The invention relates, in someaspects, to methods for producing mature microRNA in sufficientquantities for therapeutic and research applications.

The methods for efficient expression of microRNA involve the use of aprecursor microRNA molecule having a microRNA sequence in the context ofmicroRNA flanking sequences. The precursor microRNA is composed of anytype of nucleic acid based molecule capable of accommodating themicroRNA flanking sequences and the microRNA sequence. Examples ofprecursor microRNAs and the individual components of the precursor(flanking sequences and microRNA sequence) are provided herein. Theinvention, however, is not limited to the examples provided. Theinvention is based, at least in part, on the discovery of an importantcomponent of precursor microRNAs, that is, the microRNA flankingsequences. The nucleotide sequence of the precursor and its componentsmay vary widely.

In one aspect a precursor microRNA molecule is an isolated nucleic acidincluding microRNA flanking sequences and having a stem-loop structurewith a microRNA sequence incorporated therein. An “isolated molecule” isa molecule that is free of other substances with which it is ordinarilyfound in nature or in vivo systems to an extent practical andappropriate for its intended use. In particular, the molecular speciesare sufficiently free from other biological constituents of host cellsor if they are expressed in host cells they are free of the form orcontext in which they are ordinarily found in nature. For instance, anucleic acid encoding a precursor microRNA having homologous microRNAsequences and flanking sequences may ordinarily be found in a host cellin the context of the host cell genomic DNA. An isolated nucleic acidencoding a microRNA precursor may be delivered to a host cell, but isnot found in the same context of the host genomic DNA as the naturalsystem. Alternatively, an isolated nucleic acid is removed from the hostcell or present in a host cell that does not ordinarily have such anucleic acid sequence. Because an isolated molecular species of theinvention may be admixed with a pharmaceutically-acceptable carrier in apharmaceutical preparation or delivered to a host cell, the molecularspecies may comprise only a small percentage by weight of thepreparation or cell. The molecular species is nonetheless isolated inthat it has been substantially separated from the substances with whichit may be associated in living systems.

An “isolated precursor microRNA molecule” is one which is produced froma vector having a nucleic acid encoding the precursor microRNA. Thus,the precursor microRNA produced from the vector may be in a host cell orremoved from a host cell. The isolated precursor microRNA may be foundwithin a host cell that is capable of expressing the same precursor. Itis nonetheless isolated in that it is produced from a vector and, thus,is present in the cell in a greater amount than would ordinarily beexpressed in such a cell.

The term “nucleic acid” is used to mean multiple nucleotides (i.e.molecules comprising a sugar (e.g. ribose or deoxyribose) linked to aphosphate group and to an exchangeable organic base, which is either asubstituted pyrimidine (e.g. cytosine (C), thymidine (T) or uracil (U))or a substituted purine (e.g. adenine (A) or guanine (G)). The termshall also include polynucleosides (i.e. a polynucleotide minus thephosphate) and any other organic base containing polymer. Purines andpyrimidines include but are not limited to adenine, cytosine, guanine,thymidine, inosine, 5-methylcytosine, 2-aminopurine,2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, and othernaturally and non-naturally occurring nucleobases, substituted andunsubstituted aromatic moieties. Other such modifications are well knownto those of skill in the art. Thus, the term nucleic acid alsoencompasses nucleic acids with substitutions or modifications, such asin the bases and/or sugars.

“MicroRNA flanking sequence” as used herein refers to nucleotidesequences including microRNA processing elements. MicroRNA processingelements are the minimal nucleic acid sequences which contribute to theproduction of mature microRNA from precursor microRNA. Often theseelements are located within a 40 nucleotide sequence that flanks amicroRNA stem-loop structure. In some instances the microRNA processingelements are found within a stretch of nucleotide sequences of between 5and 4,000 nucleotides in length that flank a microRNA stem-loopstructure.

Thus, in some embodiments the flanking sequences are 5-4,000 nucleotidesin length. As a result, the length of the precursor molecule may be, insome instances at least about 150 nucleotides or 270 nucleotides inlength. The total length of the precursor molecule, however, may begreater or less than these values. In other embodiments the minimallength of the microRNA flanking sequence is 10, 20, 30, 40, 50, 60, 70,80, 90, 100, 150, 200 and any integer there between. In otherembodiments the maximal length of the microRNA flanking sequence is2,000, 2,100, 2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900,3,000, 3,100, 3,200, 3,300, 3,400, 3,500, 3,600, 3,700, 3,800, 3,9004,000 and any integer there between.

The microRNA flanking sequences may be native microRNA flankingsequences or artificial microRNA flanking sequences. A native microRNAflanking sequence is a nucleotide sequence that is ordinarily associatedin naturally existing systems with microRNA sequences, i.e., thesesequences are found within the genomic sequences surrounding the minimalmicroRNA hairpin in vivo. Artificial microRNA flanking sequences arenucleotides sequences that are not found to be flanking to microRNAsequences in naturally existing systems. The artificial microRNAflanking sequences may be flanking sequences found naturally in thecontext of other microRNA sequences. Alternatively they may be composedof minimal microRNA processing elements which are found within naturallyoccurring flanking sequences and inserted into other random nucleic acidsequences that do not naturally occur as flanking sequences or onlypartially occur as natural flanking sequences.

The microRNA flanking sequences within the precursor microRNA moleculemay flank one or both sides of the stem-loop structure encompassing themicroRNA sequence. Thus, one end (i.e., 5′) of the stem-loop structuremay be adjacent to a single flanking sequence and the other end (i.e.,3′) of the stem-loop structure may not be adjacent to a flankingsequence. Preferred structures have flanking sequences on both ends ofthe stem-loop structure. The flanking sequences may be directly adjacentto one or both ends of the stem-loop structure or may be connected tothe stem-loop structure through a linker, additional nucleotides orother molecules.

A “stem-loop structure” refers to a nucleic acid having a secondarystructure that includes a region of nucleotides which are known orpredicted to form a double strand (stem portion) that is linked on oneside by a region of predominantly single-stranded nucleotides (loopportion). The terms “hairpin” and “fold-back” structures are also usedherein to refer to stem-loop structures. Such structures are well knownin the art and the term is used consistently with its known meaning inthe art. The actual primary sequence of nucleotides within the stem-loopstructure is not critical to the practice of the invention as long asthe secondary structure is present. As is known in the art, thesecondary structure does not require exact base-pairing. Thus, the stemmay include one or more base mismatches. Alternatively, the base-pairingmay be exact, i.e. not include any mismatches.

In some instances the precursor microRNA molecule may include more thanone stem-loop structure. The multiple stem-loop structures may be linkedto one another through a linker, such as, for example, a nucleic acidlinker or by a microRNA flanking sequence or other molecule or somecombination thereof.

An example of a precursor microRNA is the following:

-   -   wherein X₁, and X₂ are nucleotides and wherein N₁ and N₂ are        nucleic acids of 16-28 nucleotides in length. Such a structure        is a single example. As described herein the actual nucleotide        sequence of the precursor molecule can vary significantly. In        general, X₁, and X₂ refers to the microRNA flanking sequences        and N₁ and N₂ refers to the microRNA sequence and the        corresponding sequence that is often degraded and sometimes        referred to as microRNA*.

N₁ and N₂ have at least partial complementarity. “Partialcomplementarity” when used in this context refers to at least a portionof the nucleic acid sequences that are capable of base pairing. Forinstance, in some embodiments two nucleic acid sequences that havepartial complementarity have at least 10 nucleotides that are capable ofbase pairing. In some instances, at least 15 nucleotides in eachsequence are capable of participating in a base paring interaction withone another. In other instances, the two nucleic acids are perfectlycomplementary, and thus all nucleotides in each sequence are capable ofbase pairing with a corresponding nucleotide in the other nucleic acidsequence.

A microRNA sequence is incorporated into the stem-loop structure of theprecursor microRNA molecule. As used herein, the term “microRNA” refersto any type of interfering RNA, including but not limited to, endogenousmicroRNA and artificial microRNA. Endogenous microRNA are small RNAsnaturally present in the genome which are capable of modulating theproductive utilization of mRNA. The term artificial microRNA includesany type of RNA sequence, other than endogenous microRNA, which iscapable of modulating the productive utilization of mRNA. For instance,it includes sequences previously identified as siRNA, regardless of themechanism of down-stream processing of the RNA (i.e. although siRNAs arebelieved to have a specific method of in vivo processing resulting inthe cleavage of mRNA, such sequences can be incorporated into thevectors in the context of the flanking sequences described herein). Thusa microRNA sequence is a nucleic acid composed of any one or more ofthese sequences. MicroRNA sequences have been described in publicationssuch as, Lim, et al., Genes & Development, 17, p. 991-1008 (2003), Limet al Science 299, 1540 (2003), Lee and Ambrose Science, 294, 862(2001), Lau et al., Science 294, 858-861 (2001), Lagos-Quintana et al,Current Biology, 12, 735-739 (2002), Lagos-Quintana et al, Science 294,853-857 (2001), and Lagos-Quintana et al, RNA, 9, 175-179 (2003), whichare incorporated by reference. Multiple microRNAs may also beincorporated into the precursor molecule.

The term “productive utilization of mRNA” refers to any change withinthe cell resulting in less protein accumulating from the mRNA. Forinstance a compound that interferes with translation of mRNA, would be acompound that modulates productive utilization of mRNA. Similarly, acompound that specifies the cleavage of an mRNA would be a compound thatmodulates productive utilization of that mRNA.

In some instances, the precursor microRNA includes a microRNA sequenceand a microRNA flanking sequence that are derived from the same microRNAgene and in other instances it includes a microRNA sequence and amicroRNA flanking sequence that are not derived from the same microRNAgene. The term “that is derived from the same microRNA gene” refers to anucleic acid sequence that includes both the microRNA sequence and themicroRNA flanking sequence(s) that is identical to a nucleic acid foundin nature. The term “that is not derived from the same microRNA gene”refers to a nucleic acid sequence that includes both the microRNAsequence and the microRNA flanking sequence(s) and that is not identicalto a nucleic acid found in nature. Thus, in some instances, theprecursor microRNA will include a flanking microRNA sequence that is notordinarily associated in nature with the microRNA with which it isassociated in the precursor molecule. An artificial microRNA willalways, for instance, not be derived from the same microRNA gene as theflanking sequence with which it is associated. Additionally, even if amicroRNA sequence and a microRNA flanking sequence are found within acommon gene in nature, a precursor microRNA molecule according to theinvention is said to include a microRNA sequence and a microRNA flankingsequence that are not derived from the same gene, if the structure ofthe precursor is modified in any way from that which is ordinarily foundin nature, i.e. a nucleotide is changed from a naturally occurringnucleotide or an additional nucleotide (s) or linker is inserted, etc.

In some instances the precursor microRNAs described herein do notinclude bantam microRNA sequences and flanking sequences. In otherinstances bantam microRNA sequences and/or flanking sequences areincluded within the compounds and methods of the invention.

A precursor microRNA molecule may be processed in vivo or in vitro toproduce a mature microRNA. A precursor microRNA molecule is processed ina host cell by a ribonuclease enzyme or enzymes. One example of aribonuclease enzyme which processes precursor microRNA molecules is theRNase II ribonuclease Dicer.

A mature microRNA is a functional microRNA which is capable ofmodulating or altering the productive utilization of mRNA, i.e.,regulating the expression of protein-coding genes at thepost-transcriptional level. These methods are described in more detailbelow. Mature microRNAs generally have a length of between 16 and 28nucleotides and more often between 21 and 24 nucleotides.

One advantage of the methods and products described herein is theefficient processing of microRNAs. A related advantage is the accuracyof processing. MicroRNAs are generally processed asymmetrically in vivoi.e., only the functional strand is incorporated into a ribonuceoproteincomplex-miRNP. This is true for microRNA as well as siRNAs, which areprocessed into RNA-induced protein complex-RISC. This type of processingis required for the molecule to be functional and stable. The RISC andmiRNP are similar, if not identical. Selective incorporation of thefunctional strand of microRNA, artificial microRNA or siRNA into theseprotein complexes will increase the efficacy, specificity, and stabilityof the small RNAs. The rules for selective incorporation of thefunctional strand into RISC or miRNP are not fully known. But themethods described herein allow selective incorporation of the functionalstrand into RISC or miRNP and thus result in significantly enhancedproduction of functional microRNA protein complexes.

The invention also includes vectors for producing precursor microRNAmolecules. Generally these vectors include a sequence encoding aprecursor microRNA and in vivo expression elements. The in vivoexpression elements include at least one promoter. The vector or primarytranscript is first processed to produce the stem-loop precursormolecule. The stem-loop precursor is then processed to produce themature microRNA.

One example of a vector useful for expressing the precursor microRNAs isshown in SEQ ID NO. 1. Thus the invention encompasses the nucleotidesequence of SEQ ID NO 1 as well as variants thereof.

In general, variants typically will share at least 40% nucleotideidentity with SEQ ID NO: 1, in some instances, will share at least 50%nucleotide identity; and in still other instances, will share at least60% nucleotide identity. The preferred variants have at least 70%sequence homology to SEQ ID NO: 1. More preferably the preferredvariants have at least 80% and, most preferably, at least 90% sequencehomology to SEQ ID NO: 1.

Variants with high percentage sequence homology can be identified, forexample, using stringent hybridization conditions. The term “stringentconditions”, as used herein, refers to parameters with which the art isfamiliar. More specifically, stringent conditions, as used herein, referto hybridization at 65° C. in hybridization buffer (3.5×SSC, 0.02%Ficoll, 0.02% polyvinyl pyrolidone, 0.02% bovine serum albumin, 2.5 mMNaH₂PO₄ (pH 7), 0.5% SDS, 2 mM EDTA). SSC is 0.15M sodium chloride/0.15Msodium citrate, pH 7; SDS is sodium dodecyl sulphate; and EDTA isethylenediaminetetraacetic acid. After hybridization, the membrane towhich the DNA is transferred is washed at 2×SSC at room temperature andthen at 0.1×SSC/0.1×SDS at 65° C. There are other conditions, reagents,and so forth which can be used, which result in a similar degree ofstringency.

The “in vivo expression elements” are any regulatory nucleotidesequence, such as a promoter sequence or promoter-enhancer combination,which facilitates the efficient expression of the nucleic acid toproduce the precursor microRNA. The in vivo expression element may, forexample, be a mammalian or viral promoter, such as a constitutive orinducible promoter or a tissue specific promoter. Constitutive mammalianpromoters include, but are not limited to, polymerase promoters as wellas the promoters for the following genes: hypoxanthine phosphoribosyltransferase (HPTR), adenosine deaminase, pyruvate kinase, and β-actin.Exemplary viral promoters which function constitutively in eukaryoticcells include, for example, promoters from the simian virus, papillomavirus, adenovirus, human immunodeficiency virus (HIV), Rous sarcomavirus, cytomegalovirus, the long terminal repeats (LTR) of moloneyleukemia virus and other retroviruses, and the thymidine kinase promoterof herpes simplex virus. Other constitutive promoters are known to thoseof ordinary skill in the art. The promoters useful as in vivo expressionelement of the invention also include inducible promoters. Induciblepromoters are expressed in the presence of an inducing agent. Forexample, the metallothionein promoter is induced to promotetranscription in the presence of certain metal ions. Other induciblepromoters are known to those of ordinary skill in the art.

In general, the in vivo expression element shall include, as necessary,5′ non-transcribing and 5′ non-translating sequences involved with theinitiation of transcription. They optionally include enhancer sequencesor upstream activator sequences as desired.

Vectors include, but are not limited to, plasmids, phagemids, viruses,other vehicles derived from viral or bacterial sources that have beenmanipulated by the insertion or incorporation of the nucleic acidsequences for producing the precursor microRNA, and free nucleic acidfragments which can be attached to these nucleic acid sequences. Viraland retroviral vectors are a preferred type of vector and include, butare not limited to, nucleic acid sequences from the following viruses:retroviruses, such as: Moloney murine leukemia virus; Murine stem cellvirus, Harvey murine sarcoma virus; murine mammary tumor virus; Roussarcoma virus; adenovirus; adeno-associated virus; SV40-type viruses;polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpesviruses; vaccinia viruses; polio viruses; and RNA viruses such as anyretrovirus. One can readily employ other vectors not named but known inthe art.

Viral vectors are generally based on non-cytopathic eukaryotic virusesin which non-essential genes have been replaced with the nucleic acidsequence of interest. Non-cytopathic viruses include retroviruses, thelife cycle of which involves reverse transcription of genomic viral RNAinto DNA with subsequent proviral integration into host cellular DNA.Retroviruses have been approved for human gene therapy trials.Genetically altered retroviral expression vectors have general utilityfor the high-efficiency transduction of nucleic acids in vivo. Standardprotocols for producing replication-deficient retroviruses (includingthe steps of incorporation of exogenous genetic material into a plasmid,transfection of a packaging cell lined with plasmid, production ofrecombinant retroviruses by the packaging cell line, collection of viralparticles from tissue culture media, and infection of the target cellswith viral particles) are provided in Kriegler, M., “Gene Transfer andExpression, A Laboratory Manual,” W.H. Freeman Co., New York (1990) andMurry, E. J. Ed. “Methods in Molecular Biology,” vol. 7, Humana Press,Inc., Cliffton, N.J. (1991).

The invention also encompasses host cells transfected with thesevectors. Host cells include for instance, cells and cell lines, e.g.prokaryotic (e.g., E. coli), and eukaryotic (e.g., dendritic cells, CHOcells, COS cells, yeast expression systems and recombinant baculovirusexpression in insect cells).

The precursor microRNA, and subsequently the mature functional microRNAsare useful for altering accumulation of one or more target proteins.This may be accomplished by contacting a cell with a vector capable ofexpressing a precursor microRNA as described herein. The vector producesthe microRNA transcript, which is then processed into precursor microRNAin the cell, which is then processed to produce the mature functionalmicroRNA which is capable of altering accumulation of the targetprotein. Accumulation of the protein may be effected in a number ofdifferent ways. For instance the microRNA may directly or indirectlyaffect translation or may result in cleavage of the mRNA transcript oreven effect stability of the protein being translated from the targetmRNA. MicroRNA may function through a number of different mechanisms.The methods and products of the invention are not limited to any onemechanism. The method may be performed in vitro, e.g., for studying genefunction, ex vivo or in vivo, e.g. for therapeutic purposes.

An “ex vivo” method as used herein is a method which involves isolationof a cell from a subject, manipulation of the cell outside of the body,and reimplantation of the manipulated cell into the subject. The ex vivoprocedure may be used on autologous or heterologous cells, but ispreferably used on autologous cells. In preferred embodiments, the exvivo method is performed on cells that are isolated from bodily fluidssuch as peripheral blood or bone marrow, but may be isolated from anysource of cells. When returned to the subject, the manipulated cell willbe programmed for cell death or division, depending on the treatment towhich it was exposed. Ex vivo manipulation of cells has been describedin several references in the art, including Engleman, E. G., 1997,Cytotechnology, 25:1; Van Schooten, W., et al., 1997, Molecular MedicineToday, June, 255; Steinman, R. M., 1996, Experimental Hematology, 24,849; and Gluckman, J. C., 1997, Cytokines, Cellular and MolecularTherapy, 3:187. The ex vivo activation of cells of the invention may beperformed by routine ex vivo manipulation steps known in the art. Invivo methods are also well known in the art. The invention thus isuseful for therapeutic purposes and also is useful for research purposessuch as testing in animal or in vitro models of medical, physiologicalor metabolic pathways or conditions.

The ex vivo and in vivo methods are performed on a subject. A “subject”shall mean a human or non-human mammal, including but not limited to, adog, cat, horse, cow, pig, sheep, goat, primate, rat, and mouse.

In some instances the mature microRNA is expressed at a level sufficientto cause at least a 2-fold or in some instances a 10 fold reduction inaccumulation of the target protein. The level of accumulation of atarget protein may be assessed using routine methods known to those ofskill in the art. For instance, protein may be isolated from a targetcell and quantitated using Western blot analysis or other comparablemethodologies, optionally in comparison to a control. Protein levels mayalso be assessed using reporter systems or fluorescently labeledantibodies. In other embodiments, the mature microRNA is expressed at alevel sufficient to cause at least a 2, 5, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, or 100 fold reduction in accumulation of thetarget protein. The “fold reduction” may be assessed using any parameterfor assessing a quantitative value of protein expression. For instance,a quantitative value can be determined using a label i.e. fluorescent,radioactive linked to an antibody. The value is a relative value that iscompared to a control or a known value.

Different microRNA sequences have different levels of expression ofmature microRNA and thus have different effects on target mRNA and/orprotein expression. For instance, in some cases a microRNA may beexpressed at a high level and may be very efficient such that theaccumulation of the target protein is completely or near completelyblocked. In other instances the accumulation of the target protein maybe only reduced slightly over the level that would ordinarily beexpressed in that cell at that time under those conditions in theabsence of the mature microRNA. Complete inhibition of the accumulationof the target protein is not essential for therapeutic purposes. In manycases partial or low inhibition of accumulation may produce a preferredphenotype. The actual amount that is useful will depend on theparticular cell type, the stage of differentiation, conditions to whichthe cell is exposed, the modulation of other target proteins, etc.

The microRNAs may be used to knock down gene expression in vertebratecells for gene-function studies, including target-validation studiesduring the development of new pharmaceuticals, as well as thedevelopment of human disease models and therapies, and ultimately, humangene therapies.

The methods of the invention are useful for treating any type of“disease”, “disorder” or “condition” in which it is desirable to reducethe expression or accumulation of a particular target protein(s).Diseases include, for instance, but are not limited to, cancer,infectious disease, cystic fibrosis, blood disorders, including leukemiaand lymphoma, spinal muscular dystrophy, early-onset Parkinsonism(Waisman syndrome) and X-linked mental retardation (MRX3).

The microRNAs are useful in research and therapeutics related tohematopoiesis. During hematopoiesis at least eight distinct lineages ofmature blood cells are formed as the descendents of the multipotentialhematopoietic stem cells (HSCs). Hematopoietic stem cells first arisewithin the extra-embryonic yolk sac and later theaortic-gonad-mesonephros (AGM) region of the developing embryo.Thereafter hematopoiesis normally occurs in the fetal liver and in adultbone marrow. In the adult certain stresses induce extramedullaryhematopoiesis, especially in the spleen. Hematopoiesis is sustained forlife by self-renewal of the HSCs and their continuous development intoall blood cells types. The extraordinary ability of HSCs to self-renewand differentiate was demonstrated by the repopulation of the entireblood system of a mouse by a single stem cell. Because of their abilityto self-renew and to differentiate into all blood cells, HSCs form thebasis of bone marrow transplantation for treatment of leukemias andother cancers and several nonmalignant blood cell disorders.

In order to identify microRNAs that might play roles in hematopoiesis,microRNAs were cloned from mouse bone marrow, the primary adulthematopoietic organ in vertebrates. 2180 tiny RNAs isolated from mousebone marrow were cloned and sequenced. These represented about 100unique microRNA. These hematopoietic microRNAs are listed in Table 1.Nineteen frequently cloned microRNAs shown in Table 2 were subjected tofurther analysis. These included 15 previously identified microRNAs and4 newly identified microRNAs. All but two of these microRNAs (LM3_A01-3and miR-191) were perfectly conserved in the human genome. TABLE 1 Someof the microRNAs frequently cloned from mouse bone marrow. SomemicroRNAs were represented by clones of different lengths, due toheterogeneity at the microRNA 3′ terminus. The observed size range isindicated, as is the microRNA sequence of the most abundant length. Size# of microRNA Seq. Id microRNA sequence range clones Location begin endlet-7c LM2_B07-1 (SEQ ID NO:7) 22-23 12 CONTIG_128829 246 267UGAGGUAGUAGGUUGUAUGGUU let-7g LP1_B02-8 (SEQ ID NO:8) 20-23 11CONTIG_265484 2018 2038 UGAGGUAGUAGUUUGUACAGU miR-15a B1_F06-2 (SEQ IDNO:9) 21-22 4 CONTIG_87894 853 832 UAGCAGCACAUAAUGGUUUGUG miR-16B1_D01-3 (SEQ ID NO:10) 21-25 34 CONTIG_87894 713 693UAGCAGCACGUAAAUAUUGGCG miR-19b LM1_B07-3 (SEQ ID NO:11) 21-23 9CONTIG_347954 1756 1778 UGUGCAAAUCCAUGCAAAACUGA miR-20 LM1_B03-2 (SEQ IDNO:12) 22-24 18 Hs13_10023 1387067 1387089 UAAAGUGCUUAUAGUGCAGGUAGmiR-23a LP1_B05-2 (SEQ ID NO:13) 21-23 5 CONTIG_548175 437 458AUCACAUUGCCAGGGAUUUCCA miR-27b B2_E07-1 SEQ ID NO:13) 20-23 7 Hs9_86331373759 1373779 UUCACAGUGGCUAAGUUCUGC miR-29a LM4_A03-3 (SEQ ID NO:15)22-22 20 Hs7_23805 55795 55774 UAGCACCAUCUGAAAUCGGUUA miR-30b B1_B01-2(SEQ ID NO:6) 22-23 12 CONTIG_572951 1505 1384 UGUAAACAUCCUACACUCAGCUmiR-30c B1_C07-1 (SEQ ID NO:16) 23-25 13 CONTIG_117246 3389 3412UGUAAACAUCCUACACUCUCAGCU miR-104 B1_D01-7 (SEQ ID NO:17) 21-24 37CONTIG_168444 1754 1732 UAGCUUAUCAGACUGAUGUUGAC miR-132s B1_G04-1 (SEQID NO:5) 22-23 16 Hs17_10808 2182613 2182634 CCCAUAAAGUAGAAAGCACUACmir-191 LM1_E02-3 (SEQ ID NO:18) 20-24 32 CONTIG_261531 3175 3196CAACGGAAUCCCAAAAGCAGCU miR-223 B1_E08-6 (SEQ ID NO:3) 20-24 65CONTIG_202715 440 418 UGUCAGUUUGUCAAAUACCCCAA new B1_G08-2 (SEQ IDNO:19) 22-23 7 Hs8_8395 132054 132033 UCCUGUACUGAGCUGCCCCGAG newLP1_B02-3 (SEQ ID NO:20) 21-22 7 CONTIG_7440 8193 8213UUAUAAAGCAAUGAGACUGAU new LM3_A01-3 (SEQ ID NO:21) 22-24 10CONTIG_195284 13756 13734 UGAGGUAUUAGUUUGUGCUGUUA new LM4_D05-3 (SEQ IDNO:22) 17-23 8 Hs16_19764 637009 636987 UACCACAGGGUAGAACCACGGAC

To identify microRNAs expressed at sites of hematopoiesis, we probednorthern blots of RNA isolated from different mouse tissues, includingbrain, heart, lung, liver, kidney, muscle, fetal liver, bone marrow,spleen, and thymus. Among these tissues, bone marrow, spleen, and thymusrepresent three major adult hematopoiesis sites. These organs playdifferent roles in adult hematopoiesis and comprise significantlydifferent cell types. Bone marrow, the primary hematopoietic organ inadult vertebrates, provides other secondary hematopoietic organs withcommitted progenitor cells. It consists of hematopoietic stem cells andmyeloid, erythroid and lymphoid cells at a variety of differentiationstages, although most bone marrow cells belong to myeloid and erythroidlineages. Thymus, the primary lymphoid organ, constitutes mainlyT-lymphocytes. Spleen, the secondary lymphoid organ, mainly comprisesdifferentiated reticulocytes and T and B lymphocytes. Fetal liver is theembryonic hematopoiesis site. Thus, analysis of microRNA expression inthese four tissues reveals not only the hematopoietic-specific microRNAsbut also can provide guidance as to their differential roles duringhematopoietic development. For 17 of the 19 microRNAs, expression wasdetected on the Northerns, and each of these had tissue-specificexpression patterns (Table 2). For 12 of these microRNAs, expression wasreadily detected in hematopoietic tissues. TABLE 3 Summary of tissue anddevelopmental expression of microRNAs cloned from mouse bone marrow.Northern blots of total RNA from a variety of mouse tissues, with afocus on hematopoietic tissues of different functions and developmentalstages, were probed for the indicated microRNA. Signal intensities wereranked (−, negative; +, positive; +++++, most positive) afternormalization to the U6 signal to compensate for differential sampleloading. Fetal Bone microRNA Seq. id Brain Heart Lung Liver KidneyMuscle liver marrow Spleen Thymus miR-15a B1_F06-2 − − +++ − ++ − − + +++ miR-16 B1_D01-3 + + +++ + ++ + + +++ +++ +++ miR-20 LM1_B03-2 − − + −− − + +++ ++ ++++ miR-132s B1_G04-1 − − + − − − + +++ +++ +++ miR-223B1_E08-6 − − + − − − − ++++ − − let-7c LM2_B07-1 +++ ++ ++++ − ++ +− + + + let-7g LP1_B02-8 + + +++ ++ ++ + − + + + miR-19b LM1_B07-3 − − +− − − − − − − miR-23a LP1_B05-2 − + +++ − + + − − ++ − miR-27b B2_E07-1− − ++++ − + − − − − − miR-29a LM4_A03-3 +++++ +++ ++++ +++ +++ ++ − +++++ ++ miR-30b B1_B01-2 − − + − + − − − − − miR-30c B1_C07-1 − − + − +− − − − − miR-104 B1_D01-7 − − ++++ +++ ++ + − + ++ + miR-191 LM1_E02-3− + − − − − − − − − new LP1_B02-3 − − + + − − − + + − new B1_G08-2 + + +− − − − − + − new LM3_A01-3 − − − − − − − − − − new LM4_D05-3 − − − − −− − − − −

The following five microRNAs, mir-15a, miR-16, miR-20, miR-132s andmiR-223, exhibited high and often preferential accumulation inhematopoietic tissues and were found to be regulated by cytokines.

miR-132s, the microRNA found at a breakpoint of a t(8:17) translocationassociated with an aggressive B-cell leukemia, is primarily expressed inhematopoietic tissues (with the lung being the only other tissue withappreciable expression). Significant expression of this microRNA wasseen already in E13 fetal liver. The expression of mature miR-132s inbone marrow and spleen was comparable and about 2-fold higher than thatin thymus. Interestingly, accumulation of the presumed miR-132 precursorwas high, and the ratio of mature and precursor RNAs varied in differenttissues, suggesting regulation at the level of Dicer processing.

miR-20 was expressed in a similar pattern as miR-132s, though there wassubstantially higher accumulation of this microRNA in the thymuscompared to the spleen.

miR-223 had the most striking tissue specificity of any of the microRNAexamined. It was very strongly expressed in bone marrow, and wasdetectable in spleen but essentially negative in thymus, E13 fetal liverand all other mouse tissues, except the lung.

miR-16 and miR-15a are the two microRNAs that derive from band 13q13.3of the human chromosome 13, the site of the most common structuralaberrations in both mantle cell lymphoma and B-cell chronic lymphocyticleukemia. Their loci are within 130 bp of each other, suggesting thatthey might be transcribed and processed from a single primarytranscript. Consistent with this idea, they have similar expressionpatterns, except that miR-16 appears to accumulate to much higherlevels. Expression is high in the adult hematopoietic tissues, althoughthese microRNAs, particularly miR-16, can be readily detected in othertissues.

Two of these microRNAs, mir-15a and miR-16, are within band 13q13—aregion of human chromosome 13 that is thought to harbor a lymphoidregulatory locus because it is the site of the most common structuralaberrations in both mantle cell lymphoma and B-cell chronic lymphocyticleukemia. Another one of the hematopoietic microRNA genes, mir-132, mapsto the breakpoint junction of a t(8;17) translocation that has beenlinked to an aggressive B cell leukemia. In this translocation, atruncated MYC gene is fused to the promoter and 5′ portion of themir-132 gene. This expression data, together with chromosomalaberrations associated with human leukemias, implicate microRNAs in thedevelopmental decisions of hematopoiesis. Thus, the microRNAs are usefulin therapeutic protocols related to hematopoeitic disorders includingleukemias and lymphomas.

Cancers include but are not limited to biliary tract cancer; bladdercancer; breast cancer; brain cancer including glioblastomas andmedulloblastomas; cervical cancer; choriocarcinoma; colon cancerincluding colorectal carcinomas; endometrial cancer; esophageal cancer;gastric cancer; head and neck cancer; hematological neoplasms includingacute lymphocytic and myelogenous leukemia, multiple myeloma,AIDS-associated leukemias and adult T-cell leukemia lymphoma;intraepithelial neoplasms including Bowen's disease and Paget's disease;liver cancer; lung cancer including small cell lung cancer and non-smallcell lung cancer; lymphomas including Hodgkin's disease and lymphocyticlymphomas; neuroblastomas; oral cancer including squamous cellcarcinoma; osteosarcomas; ovarian cancer including those arising fromepithelial cells, stromal cells, germ cells and mesenchymal cells;pancreatic cancer; prostate cancer; rectal cancer; sarcomas includingleiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, synovialsarcoma and osteosarcoma; skin cancer including melanomas, Kaposi'ssarcoma, basocellular cancer, and squamous cell cancer; testicularcancer including germinal tumors such as seminoma, non-seminoma(teratomas, choriocarcinomas), stromal tumors, and germ cell tumors;thyroid cancer including thyroid adenocarcinoma and medullar carcinoma;transitional cancer and renal cancer including adenocarcinoma and Wilmstumor.

An infectious disease, as used herein, is a disease arising from thepresence of a foreign microorganism in the body. A microbial antigen, asused herein, is an antigen of a microorganism. Microorganisms includebut are not limited to, infectious virus, infectious bacteria, andinfectious fungi.

Examples of infectious virus include but are not limited to:Retroviridae (e.g. human immunodeficiency viruses, such as HIV-1 (alsoreferred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III; and otherisolates, such as HIV-LP; Picornaviridae (e.g. polio viruses, hepatitisA virus; enteroviruses, human Coxsackie viruses, rhinoviruses,echoviruses); Calciviridae (e.g. strains that cause gastroenteritis);Togaviridae (e.g. equine encephalitis viruses, rubella viruses);Flaviridae (e.g. dengue viruses, encephalitis viruses, yellow feverviruses); Coronoviridae (e.g. coronaviruses); Rhabdoviradae (e.g.vesicular stomatitis viruses, rabies viruses); Coronaviridae (e.g.coronaviruses); Rhabdoviridae (e.g. vesicular stomatitis viruses, rabiesviruses); Filoviridae (e.g. ebola viruses); Paramyxoviridae (e.g.parainfluenza viruses, mumps virus, measles virus, respiratory syncytialvirus); Orthomyxoviridae (e.g. influenza viruses); Bungaviridae (e.g.Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g. reoviruses,orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis Bvirus); Parvovirida (parvoviruses); Papovaviridae (papilloma viruses,polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae(herpes simplex virus (HSV) 1 and 2, varicella zoster virus,cytomegalovirus (CMV), herpes virus; Poxyiridae (variola viruses,vaccinia viruses, pox viruses); and Iridoviridae (e.g. African swinefever virus); and unclassified viruses (e.g. the etiological agents ofSpongiform encephalopathies, the agent of delta hepatitis (thought to bea defective satellite of hepatitis B virus), the agents of non-A, non-Bhepatitis (class 1=internally transmitted; class 2=parenterallytransmitted (i.e. Hepatitis C); Norwalk and related viruses, andastroviruses).

Examples of infectious bacteria include but are not limited to:Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia,Mycobacteria sps (e.g. M. tuberculosis, M. avium, M. intracellulare, M.kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae,Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes(Group A Streptococcus), Streptococcus agalactiae (Group BStreptococcus), Streptococcus (viridans group), Streptococcus faecalis,Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcuspneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilusinfluenzae, Bacillus antracis, corynebacterium diphtheriae,corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridiumperfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiellapneumoniae, Pasturella multocida, Bacteroides sp., Fusobacteriumnucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponemapertenue, Leptospira, Rickettsia, and Actinomyces israelli.

Examples of infectious fungi include: Cryptococcus neoformans,Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis,Chlamydia trachomatis, Candida albicans. Other infectious organisms(i.e., protists) include: Plasmodium such as Plasmodium falciparum,Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax andToxoplasma gondii.

In one aspect, the invention provides a method of administering any ofthe compositions described herein to a subject. When administered, thecompositions are applied in a therapeutically effective,pharmaceutically acceptable amount as a pharmaceutically acceptableformulation. As used herein, the term “pharmaceutically acceptable” isgiven its ordinary meaning. Pharmaceutically acceptable compounds aregenerally compatible with other materials of the formulation and are notgenerally deleterious to the subject. Any of the compositions of thepresent invention may be administered to the subject in atherapeutically effective dose. A “therapeutically effective” or an“effective” as used herein means that amount necessary to delay theonset of, inhibit the progression of, halt altogether the onset orprogression of, diagnose a particular condition being treated, orotherwise achieve a medically desirable result, i.e., that amount whichis capable of at least partially preventing, reversing, reducing,decreasing, ameliorating, or otherwise suppressing the particularcondition being treated. A therapeutically effective amount can bedetermined on an individual basis and will be based, at least in part,on consideration of the species of mammal, the mammal's age, sex, size,and health; the compound and/or composition used, the type of deliverysystem used; the time of administration relative to the severity of thedisease; and whether a single, multiple, or controlled-release doseregiment is employed. A therapeutically effective amount can bedetermined by one of ordinary skill in the art employing such factorsand using no more than routine experimentation.

The terms “treat,” “treated,” “treating,” and the like, when usedherein, refer to administration of the systems and methods of theinvention to a subject, which may, for example, increase the resistanceof the subject to development or further development of cancers, toadministration of the composition in order to eliminate or at leastcontrol a cancer or a infectious disease, and/or to reduce the severityof the cancer or infectious disease. When administered to a subject,effective amounts will depend on the particular condition being treatedand the desired outcome. A therapeutically effective dose may bedetermined by those of ordinary skill in the art, for instance,employing factors such as those further described below and using nomore than routine experimentation.

In administering the systems and methods of the invention to a subject,dosing amounts, dosing schedules, routes of administration, and the likemay be selected so as to affect known activities of these systems andmethods. Dosage may be adjusted appropriately to achieve desired druglevels, local or systemic, depending upon the mode of administration.The doses may be given in one or several administrations per day. As oneexample, if daily doses are required, daily doses may be from about 0.01mg/kg/day to about 1000 mg/kg/day, and in some embodiments, from about0.1 to about 100 mg/kg/day or from about 1 mg/kg/day to about 10mg/kg/day. Parental administration, in some cases, may be from one toseveral orders of magnitude lower dose per day, as compared to oraldoses. For example, the dosage of an active compound when parentallyadministered may be between about 0.1 micrograms/kg/day to about 10mg/kg/day, and in some embodiments, from about 1 microgram/kg/day toabout 1 mg/kg/day or from about 0.01 mg/kg/day to about 0.1 mg/kg/day.

In some embodiments, the concentration of the active compound(s), ifadministered systemically, is at a dose of about 1.0 mg to about 2000 mgfor an adult of 70 kg body weight, per day. In other embodiments, thedose is about 10 mg to about 1000 mg/70 kg/day. In yet otherembodiments, the dose is about 100 mg to about 500 mg/70 kg/day.Preferably, the concentration, if applied topically, is about 0.1 mg toabout 500 mg/gm of ointment or other base, more preferably about 1.0 mgto about 100 mg/gm of base, and most preferably, about 30 mg to about 70mg/gm of base. The specific concentration partially depends upon theparticular composition used, as some are more effective than others. Thedosage concentration of the composition actually administered isdependent at least in part upon the particular physiological responsebeing treated, the final concentration of composition that is desired atthe site of action, the method of administration, the efficacy of theparticular composition, the longevity of the particular composition, andthe timing of administration relative to the severity of the disease.Preferably, the dosage form is such that it does not substantiallydeleteriously effect the mammal. The dosage can be determined by one ofordinary skill in the art employing such factors and using no more thanroutine experimentation.

In the event that the response of a particular subject is insufficientat such doses, even higher doses (or effectively higher doses by adifferent, more localized delivery route) may be employed to the extentthat subject tolerance permits. Multiple doses per day are alsocontemplated in some cases to achieve appropriate systemic levels withinthe subject or within the active site of the subject. In some cases,dosing amounts, dosing schedules, routes of administration, and the likemay be selected as described herein, whereby therapeutically effectivelevels for the treatment of cancer are provided.

In certain embodiments where cancers are being treated, a composition ofthe invention may be administered to a subject who has a family historyof cancer, or to a subject who has a genetic predisposition for cancer.In other embodiments, the composition is administered to a subject whohas reached a particular age, or to a subject more likely to get cancer.In yet other embodiments, the compositions is administered to subjectswho exhibit symptoms of cancer (e.g., early or advanced). In still otherembodiments, the composition may be administered to a subject as apreventive measure. In some embodiments, the inventive composition maybe administered to a subject based on demographics or epidemiologicalstudies, or to a subject in a particular field or career.

Administration of a composition of the invention to a subject may beaccomplished by any medically acceptable method which allows thecomposition to reach its target. The particular mode selected willdepend of course, upon factors such as those previously described, forexample, the particular composition, the severity of the state of thesubject being treated, the dosage required for therapeutic efficacy,etc. As used herein, a “medically acceptable” mode of treatment is amode able to produce effective levels of the active compound(s) of thecomposition within the subject without causing clinically unacceptableadverse effects.

Any medically acceptable method may be used to administer a compositionto the subject. The administration may be localized (i.e., to aparticular region, physiological system, tissue, organ, or cell type) orsystemic, depending on the condition being treated. For example, thecomposition may be administered orally, vaginally, rectally, buccally,pulmonary, topically, nasally, transdermally, through parenteralinjection or implantation, via surgical administration, or any othermethod of administration where suitable access to a target is achieved.Examples of parenteral modalities that can be used with the inventioninclude intravenous, intradermal, subcutaneous, intracavity,intramuscular, intraperitoneal, epidural, or intrathecal. Examples ofimplantation modalities include any implantable or injectable drugdelivery system. Oral administration may be preferred in someembodiments because of the convenience to the subject as well as thedosing schedule. Compositions suitable for oral administration may bepresented as discrete units such as hard or soft capsules, pills,cachettes, tablets, troches, or lozenges, each containing apredetermined amount of the active compound. Other oral compositionssuitable for use with the invention include solutions or suspensions inaqueous or non-aqueous liquids such as a syrup, an elixir, or anemulsion. In another set of embodiments, the composition may be used tofortify a food or a beverage.

Injections can be e.g., intravenous, intradermal, subcutaneous,intramuscular, or interperitoneal. The composition can be injectedinterdermally for treatment or prevention of infectious disease, forexample. In some embodiments, the injections can be given at multiplelocations. Implantation includes inserting implantable drug deliverysystems, e.g., microspheres, hydrogels, polymeric reservoirs,cholesterol matrixes, polymeric systems, e.g., matrix erosion and/ordiffusion systems and non-polymeric systems, e.g., compressed, fused, orpartially-fused pellets. Inhalation includes administering thecomposition with an aerosol in an inhaler, either alone or attached to acarrier that can be absorbed. For systemic administration, it may bepreferred that the composition is encapsulated in liposomes.

In general, the compositions of the invention may be delivered using abioerodible implant by way of diffusion, or more preferably, bydegradation of the polymeric matrix. Exemplary synthetic polymers whichcan be used to form the biodegradable delivery system include:polyamides, polycarbonates, polyalkylenes, polyalkylene glycols,polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols,polyvinyl ethers, polyvinyl esters, poly-vinyl halides,polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes andco-polymers thereof, alkyl cellulose, hydroxyalkyl celluloses, celluloseethers, cellulose esters, nitro celluloses, polymers of acrylic andmethacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropylcellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methylcellulose, cellulose acetate, cellulose propionate, cellulose acetatebutyrate, cellulose acetate phthalate, carboxylethyl cellulose,cellulose triacetate, cellulose sulphate sodium salt, poly(methylmethacrylate), poly(ethyl methacrylate), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), poly(octadecyl acrylate), polyethylene, polypropylene,poly(ethylene glycol), poly(ethylene oxide), poly(ethyleneterephthalate), poly(vinyl alcohols), polyvinyl acetate, poly vinylchloride, polystyrene, polyvinylpyrrolidone, and polymers of lactic acidand glycolic acid, polyanhydrides, poly(ortho)esters, poly(butic acid),poly(valeric acid), and poly(lactide-cocaprolactone), and naturalpolymers such as alginate and other polysaccharides including dextranand cellulose, collagen, chemical derivatives thereof (substitutions,additions of chemical groups, for example, alkyl, alkylene,hydroxylations, oxidations, and other modifications routinely made bythose skilled in the art), albumin and other hydrophilic proteins, zeinand other prolamines and hydrophobic proteins, copolymers and mixturesthereof. In general, these materials degrade either by enzymatichydrolysis or exposure to water in vivo, by surface or bulk erosion.Examples of non-biodegradable polymers include ethylene vinyl acetate,poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.

Bioadhesive polymers of particular interest include bioerodiblehydrogels described by H. S. Sawhney, C. P. Pathak and J. A. Hubell inMacromolecules, (1993) 26:581-587, the teachings of which areincorporated herein, polyhyaluronic acids, casein, gelatin, glutin,polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methylmethacrylates), poly(ethyl methacrylates), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), and poly(octadecyl acrylate).

In certain embodiments of the invention, the administration of thecomposition of the invention may be designed so as to result insequential exposures to the composition over a certain time period, forexample, hours, days, weeks, months or years. This may be accomplished,for example, by repeated administrations of a composition of theinvention by one of the methods described above, or by a sustained orcontrolled release delivery system in which the composition is deliveredover a prolonged period without repeated administrations. Administrationof the composition using such a delivery system may be, for example, byoral dosage forms, bolus injections, transdermal patches or subcutaneousimplants. Maintaining a substantially constant concentration of thecomposition may be preferred in some cases.

Other delivery systems suitable for use with the present inventioninclude time-release, delayed release, sustained release, or controlledrelease delivery systems. Such systems may avoid repeatedadministrations in many cases, increasing convenience to the subject andthe physician. Many types of release delivery systems are available andknown to those of ordinary skill in the art. They include, for example,polymer-based systems such as polylactic and/or polyglycolic acids,polyanhydrides, polycaprolactones, copolyoxalates, polyesteramides,polyorthoesters, polyhydroxybutyric acid, and/or combinations of these.Microcapsules of the foregoing polymers containing drugs are describedin, for example, U.S. Pat. No. 5,075,109. Other examples includenonpolymer systems that are lipid-based including sterols such ascholesterol, cholesterol esters, and fatty acids or neutral fats such asmono-, di- and triglycerides; hydrogel release systems; liposome-basedsystems; phospholipid based-systems; silastic systems; peptide basedsystems; wax coatings; compressed tablets using conventional binders andexcipients; or partially fused implants. Specific examples include, butare not limited to, erosional systems in which the composition iscontained in a form within a matrix (for example, as described in U.S.Pat. Nos. 4,452,775, 4,675,189, 5,736,152, 4,667,013, 4,748,034 and5,239,660), or diffusional systems in which an active component controlsthe release rate (for example, as described in U.S. Pat. Nos. 3,832,253,3,854,480, 5,133,974 and 5,407,686). The formulation may be as, forexample, microspheres, hydrogels, polymeric reservoirs, cholesterolmatrices, or polymeric systems. In some embodiments, the system mayallow sustained or controlled release of the composition to occur, forexample, through control of the diffusion or erosion/degradation rate ofthe formulation containing the composition. In addition, a pump-basedhardware delivery system may be used to deliver one or more embodimentsof the invention.

Examples of systems in which release occurs in bursts includes, e.g.,systems in which the composition is entrapped in liposomes which areencapsulated in a polymer matrix, the liposomes being sensitive tospecific stimuli, e.g., temperature, pH, light or a degrading enzyme andsystems in which the composition is encapsulated by an ionically-coatedmicrocapsule with a microcapsule core degrading enzyme. Examples ofsystems in which release of the inhibitor is gradual and continuousinclude, e.g., erosional systems in which the composition is containedin a form within a matrix and effusional systems in which thecomposition permeates at a controlled rate, e.g., through a polymer.Such sustained release systems can be e.g., in the form of pellets, orcapsules.

Use of a long-term release implant may be particularly suitable in someembodiments of the invention. “Long-term release,” as used herein, meansthat the implant containing the composition is constructed and arrangedto deliver therapeutically effective levels of the composition for atleast 30 or 45 days, and preferably at least 60 or 90 days, or evenlonger in some cases. Long-term release implants are well known to thoseof ordinary skill in the art, and include some of the release systemsdescribed above.

In some embodiments, the compositions of the invention may includepharmaceutically acceptable carriers with formulation ingredients suchas salts, carriers, buffering agents, emulsifiers, diluents, excipients,chelating agents, fillers, drying agents, antioxidants, antimicrobials,preservatives, binding agents, bulking agents, silicas, solubilizers, orstabilizers that may be used with the active compound. For example, ifthe formulation is a liquid, the carrier may be a solvent, partialsolvent, or non-solvent, and may be aqueous or organically based.Examples of suitable formulation ingredients include diluents such ascalcium carbonate, sodium carbonate, lactose, kaolin, calcium phosphate,or sodium phosphate; granulating and disintegrating agents such as cornstarch or algenic acid; binding agents such as starch, gelatin oracacia; lubricating agents such as magnesium stearate, stearic acid, ortalc; time-delay materials such as glycerol monostearate or glyceroldistearate; suspending agents such as sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose, sodium alginate,polyvinylpyrrolidone; dispersing or wetting agents such as lecithin orother naturally-occurring phosphatides; thickening agents such as cetylalcohol or beeswax; buffering agents such as acetic acid and saltsthereof, citric acid and salts thereof, boric acid and salts thereof, orphosphoric acid and salts thereof; or preservatives such as benzalkoniumchloride, chlorobutanol, parabens, or thimerosal. Suitable carrierconcentrations can be determined by those of ordinary skill in the art,using no more than routine experimentation. The compositions of theinvention may be formulated into preparations in solid, semi-solid,liquid or gaseous forms such as tablets, capsules, elixirs, powders,granules, ointments, solutions, depositories, inhalants or injectables.Those of ordinary skill in the art will know of other suitableformulation ingredients, or will be able to ascertain such, using onlyroutine experimentation.

Preparations include sterile aqueous or nonaqueous solutions,suspensions and emulsions, which can be isotonic with the blood of thesubject in certain embodiments. Examples of nonaqueous solvents arepolypropylene glycol, polyethylene glycol, vegetable oil such as oliveoil, sesame oil, coconut oil, arachis oil, peanut oil, mineral oil,injectable organic esters such as ethyl oleate, or fixed oils includingsynthetic mono or di-glycerides. Aqueous carriers include water,alcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media. Parenteral vehicles include sodium chloridesolution, 1,3-butandiol, Ringer's dextrose, dextrose and sodiumchloride, lactated Ringer's or fixed oils. Intravenous vehicles includefluid and nutrient replenishers, electrolyte replenishers (such as thosebased on Ringer's dextrose), and the like. Preservatives and otheradditives may also be present such as, for example, antimicrobials,antioxidants, chelating agents and inert gases and the like. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil may beemployed including synthetic mono- or di-glycerides. In addition, fattyacids such as oleic acid may be used in the preparation of injectables.Carrier formulation suitable for oral, subcutaneous, intravenous,intramuscular, etc. administrations can be found in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa. Those of skillin the art can readily determine the various parameters for preparingand formulating the compositions of the invention without resort toundue experimentation.

In some embodiments, the present invention includes the step of forminga composition of the invention by bringing an active compound intoassociation or contact with a suitable carrier, which may constitute oneor more accessory ingredients. The final compositions may be prepared byany suitable technique, for example, by uniformly and intimatelybringing the composition into association with a liquid carrier, afinely divided solid carrier or both, optionally with one or moreformulation ingredients as previously described, and then, if necessary,shaping the product.

In some embodiments, the compositions of the present invention may bepresent as pharmaceutically acceptable salts. The term “pharmaceuticallyacceptable salts” includes salts of the composition, prepared incombination with, for example, acids or bases, depending on theparticular compounds found within the composition and the treatmentmodality desired. Pharmaceutically acceptable salts can be prepared asalkaline metal salts, such as lithium, sodium, or potassium salts; or asalkaline earth salts, such as beryllium, magnesium or calcium salts.Examples of suitable bases that may be used to form salts includeammonium, or mineral bases such as sodium hydroxide, lithium hydroxide,potassium hydroxide, calcium hydroxide, magnesium hydroxide, and thelike. Examples of suitable acids that may be used to form salts includeinorganic or mineral acids such as hydrochloric, hydrobromic,hydroiodic, hydrofluoric, nitric, carbonic, monohydrogencarbonic,phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, phosphorous acids and the like. Other suitableacids include organic acids, for example, acetic, propionic, isobutyric,maleic, malonic, benzoic, succinic, suberic, fumaric, mandelic,phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric,methanesulfonic, glucuronic, galacturonic, salicylic, formic,naphthalene-2-sulfonic, and the like. Still other suitable acids includeamino acids such as arginate, aspartate, glutamate, and the like.

The invention also includes methods for quantitating a level ofprecursor microRNA expression. The method involves incorporating aprecursor microRNA into a reporter system, transfecting a host cell withthe reporter system, and detecting expression of a reporter gene productto quantitate the level of precursor microRNA expression. In someembodiments the reporter system includes a firefly luciferase reportergene.

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

EXAMPLES

Methods

Retroviral Constructs for microRNA expression. We have developed aretroviral vector using the murine stem cell virus (Clonetech) backbone(FIG. 1 a). A pol III expression cassette, which consists of the humanH1 promoter (P_(H1)), the microRNA hairpin and a polyT terminationsequence (T5), was placed after the viral 5′LTR resulting in a singlecopy (SC) of the microRNA, or in the viral 3′LTR whereby viralreplication resulted in a double copy (DC) of the microRNA gene (FIG. 1a). Table of miRNA sequences miR-223 (SEQ ID NO:3) B1_E08-6UGUCAGUUUGUCAAAUACCCCAA miR-181 (SEQ ID NO:4) LM3_A05-1AACAUUCAACGCUGUCGGUGA miR-132s (SEQ ID NO:5) B1_G04-1CCCAUAAAGUAGAAAGCACUAC miR-30 (SEQ ID NO:6) B1_B01-2UGUAAACAUCCUACACUCAGCU

Construction of precursor microRNA with flanking sequences. We amplifiedmiR-223 gene segments from mouse genomic DNA with lengths indicated.Each fragment was designed to give rise to a transcript containing themiR-223 minimal hairpin (67 nt) and either 0-, 10-, 20-, 40-, 60-,80-nucleotide flanking sequences on both sides of the hairpin. microRNAconstructs miR-223, miR-181 and miR-132, each designed to producetranscripts of 271-273 nucleotides in length, were also made. Theseconstructs were each cloned into the H1 expression cassette of thedouble-copy retroviral construct (FIG. 1 a). The constructs weretransiently transfected into 293T cells and expressed.

Ectopic expression of microRNAs. MicroRNAs were transiently expressed bytransfecting double-copy miRNA constructs into a mammalian cell line,i.e. 293T cells. Or, double-copy miRNA constructs were transfected intoa retroviral packaging cell line to generate retrovirus, and miRNAs werestably expressed by tranducing miRNA virus into mammalian cells, suchas, NIH3T3 or hematopoietic stem/progenitor cells. Expression ofcandidate microRNA loci was examined using Northern blots andradiolabeled DNA probes. To maintain hybridization specificity withoutvarying hybridization or washing conditions, the length of probes fordifferent sequences was adjusted so that the predicted meltingtemperatures of the microRNA-probe duplexes did not exceed 60° C. Probesnot corresponding to the entire microRNA sequence were designed tohybridize to the 3′ region of the microRNA, which is most divergentamong related microRNA sequences. The radiolabeled probes against themiRNA, or the miRNA* (the small RNA processed from the opposite arm ofthe miRNA stem-loop structure) were used to detect corresponding matureRNAs and unprocessed precursors. Ethidium staining of 5S RNA served as aloading control.

Luciferase reporter system for detection of miRNA-mediated translationalrepression. The firefly luciferase gene (Promega) was fused to a mutatedC. elegans lin-413′UTR (lin-413′UTR-delta, Genebank accession no,AF195610) in which an 80-nt segment containing both let-7 complementarysites (green) was deleted, and replaced with a miR-223 perfectcomplementary site (FIG. 5 a). In a control reporter, the Renillaluciferase gene (Promega) was fused to lin-413′UTR-delta. All luciferasegenes were under the control of the viral LTR promotor. To detecttranslational repression of the luciferase gene, virus was produced bytransfecting the constructs into the BOSC23 viral packaging cell line(Pear et al., 1993) and titred by infecting NIH3T3 cells, analyzing CD4expression using FACS. NIH3T3 cells stably expressing miR-223 or othermiRNAs were infected with equal titer of fLuc-lin41UTRdelta+miR-223target or fLuc-lin41UTRdelta−miR-223 target virus along with controlvirus rLuc-lin41UTRdelta. Four days after infection, luciferase activitywas measured using the luciferase assay (Promega). Firefly luciferaseactivity was normalized using Renilla luciferase activity.

Construction and expression of artificial microRNA/SiRNA ArtificialmicroRNA (A_miRNAs or siRNAs) were designed to target 21-nucleotideunique sequences in the firefly luciferase gene. These constructs werecloned into the B1_C02-3 microRNA template in a retroviral construct asindicated in FIG. 7. Expression of siRNAs was first tested in 293T cellsby transient transfection. Radiolabeled probes against SiLuc311 orSiLuc1276, and their complement strands, SiLuc311* or SiLuc1276*respectively, were used to detect corresponding mature A_miRNAs/siRNAsand unprocessed precursors on Northern blots. Ethidium staining of 5SRNA served as a loading control. Stable cell lines expressing individualA_miRNA/siRNAs were generated by viral infection and FACS enrichment ofcells expressing green fluorescent protein (GFP), a reporter for viralinfection (Liu et al., 2000). A_miRNA/siRNAs expressing cell lines werethen infected with firefly Luciferase virus along with control RenillaLuciferase virus. Four days after infection, luciferase activity wasmeasured using the luciferase assay. Firefly luciferase activity wasnormalized using Renilla luciferase activity.

Inducible expression of B1_C02-3 microRNA from a Polymerase II promotor.A 500 base pair B1_C02-3 microRNA gene, with predicted B1_C02-3 microRNAsequence in the center, was amplified from mouse genomic DNA. This wascloned into the TetOn inducible expression vector (Clontech). Atetracycline-inducible cell line was generated using this vector, andinduced at various doxycycline concentrations. A radiolabeled probeagainst B1_C02-3 was used to detect the mature microRNA and unprocessedprecursor, using Northern blot analysis. Ethidium staining of 5S RNAserved as a loading control.

Expression of microRNAs in developmental hematopoietic organs. Todetermine whether microRNAs are components of the molecular machinerythat regulate mouse hematopoiesis, we cloned over 100 microRNAs frommouse bone marrow and uncovered three microRNAs: miR-181, miR-223 andmiR-132, that are differentially or preferentially expressed inhematopoietic tissues and are thus considered hematopoietic-specific.

Expression of microRNAs in during hematopoietic lineage commitment. Totest whether microRNAs can regulate hematopoietic lineagedifferentiation, the effect of microRNA expression in bone marrowprogenitor cells was examined. Lineage-negative bone marrow cells wereisolated and infected with control retroviral construct (vector), orretroviral constructs expressing hematopoietic microRNAs, miR-30,miR-132s, or miR-181 and miR-223. All constructs contained a GFPreporter to indicate virally infected cells. Infected hematopoieticprogenitor cells were seeded onto S17 stromal cells, and cultured inmedium containing IL-3, IL-6, IL-7, and stem cell factor. For eachinfection, twelve culture replicates were conducted. Cells were fed withfresh growth medium every five days. After 10 days of culture, bothsuspended and adherent cells were harvested and stained with theindicated lineage markers. Virally infected cells (GFP-positive cells)were analyzed for the lineage profiles using FACS. In all cases, morethan 50% of the cells were GFP positive at the time of analysis.Hematopoietic precursor cells were infected with vectors that express acontrol vector (no microRNA), miR-30, or the hematopoietic microRNAs,miR-1{circumflex over (8)}1, miR-132s or miR-223. miR-30 which wascloned from bone marrow but only detectable in lung and kidney onNorthern was used as negative control.

Results

1. Expression of Endogenous microRNAs

Ectopic expression of microRNAs using short miRNA hairpins. Theexpression of ˜70 nt-microRNA hairpins (miR-132s) using the H1expression cassette of the “double-copy” retroviral constructs comparedwith the “single-copy” constructs was determined using Northern blotanalysis. This “double-copy” (DC) configuration, provided robust andconsistent expression of the microRNA hairpin precursors. Although thetranscribed microRNA hairpin precursors (band depicted with arrow inFIG. 1 b) were in abundance, mature microRNA products were not detectedin 293T, NIH3T3 or bone marrow cells (FIG. 1 b). The reduction of thehairpins to the minimal-length miR-223 did not detectably improve theefficiency of microRNA processing (FIG. 1 c). The substitution of aperfect match microRNA complementary strand miR-223-Si and miR-132s-Sialso did not significantly improve the efficiency of microRNA processing(FIG. 1 c.). These results demonstrated that short miRNA hairpins, whileeffectively transcribed in variety cell types using the double-copyexpression constructs, cannot be effectively processed into maturemiRNAs of endogenous forms. Changing the miRNA hairpin into a siRNA-likestem-loop (i.e., from the normally non-perfectly paired stem-loop to aperfectly paired stem-loop), did not significantly improve the miRNAprocessing efficiency. The resulting small RNA products, when detectedare typically different from the endogenous miRNAs in their size andheterogeneity pattern (FIG. 1 c). All these results suggest thatadditional elements, beside the hairpin itself, are required for propermiRNA expression and processing, and that additional elements might belocated within a larger miRNA genes that contains the miRNA hairpin.

Expression of microRNA+Flanking Sequences.

We hypothesized that elements required for miRNA maturation might becontained in the genomic sequences flanking a predicted miRNA hairpin.To test this, we amplified miR-223 genes of different length, whichcontained the miR-223 minimal hairpin and the indicated length ofgenomic sequences flanking the predicted miRNA-223 hairpin (FIG. 2 a).These gene segments were cloned into the H1 expression cassette of thedouble-copy MSCV constructs (FIG. 1 a). Northern analysis of thesemiR-223 expression revealed that miR-223 genes at least 137 nucleotideslong could be effectively processed into the hairpin precursor (FIG. 2b, arrow, P) and mature miR-223 (FIG. 2 b, arrow, miR). The maturedmiR-223 RNAs have the sizes and heterogeneity pattern similar to theendogenous bone marrow miR-223 RNAs. Interestingly, the smaller miR-223genes resulted accumulation of miR-223 gene transcripts that were notprocessed into the hairpin precursor and mature miR-223 (FIG. 2 b).

Further analysis of the cytoplasmic (FIG. 2 c) and nuclear (FIG. 2 d)localization of miR-223 transcripts and processed products revealed thatmiRNA processing was not limited by nuclear export of miR-223transcripts. Abundant miR-223 transcripts (miR-223-67Si tomiR-223_(—)107) were present in the cytoplasm but were not processedinto miR-223 hairpin precursor and mature miRNA (FIG. 2 c). This resultdemonstrates that the Dicer enzyme (a cytoplasmic enzyme that is partlyresponsible for miRNA processing) cannot directly process these longerhairpins, despite their presence in the cytoplasm. When the longermiR-223 genes were used (miR-223_(—)137 to miR-223_(—)500), maturemiR-223 can be readily detected on the northern blot (FIG. 2 b).Interestingly, a band of the size of the predicted hairpin precursor ofmiR-223 was also observed in these lanes (FIG. 2 b, P). Furthermore,mature miR-223 and its hairpin precursor can only be seen in cytoplasmfraction, but not in the nuclear fraction. Take together, these resultssuggest that a pre-Dicer processing step is be required to generate themiRNA hairpin precursor that can be recognized by Dicer. The flankingsequence of miRNA hairpin gene is essential for this non-Dicerprocessing step.

Based on the example of miR-223 gene expression (FIG. 3), 40 nt orlonger flanking sequence on one or both sides of the miR-223 hairpinprecursor is required for miR-223 expression and maturation. The lowerlimit of the length of the flanking sequences may vary from one miRNAgene to another. Thus, to ensure the flanking sequences contain properprocessing signal, we choose to express miRNA genes with 125 nt flankingsequences amplified from predicted miRNA genomic loci. Amplified miRNAgenes were place into the H1 expression cassette of the double-copyretroviral construct. We were able to express miR-223, miR-181 andmiR-132s (FIG. 3 a-c), as well as 10 other miRNA tested (data notshown). Over-expression of a hematopoietic miRNA can also be achieved inhematopoietic progenitor cells, where the endogeneous miRNA is alsopresent (FIG. 3 c).

We also compared the expression of miR-30 when expressed from a shorterhairpin (71 nt) or a longer (272 nt) miRNA gene. The longer miR-30 genecan be efficiently expressed and processed into the hairpin precursorand mature miR-30 (FIG. 4 a). While trace amount of mature miR-30 isprocessed from a shorter hairpin (71 nt), its processing is veryinefficient. Moreover, we noted that using miRNA flanking-sequence tofacilitate miRNA expression not only increased miRNA processingefficient but also helped to maintain asymmetric miRNA expression. Forexample, only the miR-30 strand but not the miR-30* strand can bedetected when miR-30 was expressed from the longer (272 nt) precursor(FIG. 4). In contrast, others have shown that both miR-30 and miR-30*strand were detected when miR-30 was expressed from a shorter hairpinprecursor placed in the context of heterogenous mRNA transcripts (Zenget al. Mol. Cell, 9:1327-1333). Maintaining asymmetrical miRNAprocessing may be critical for ensuring the specificity of miRNAregulation in vivo.

Ectopically Expressed miRNAs Repress Reporter Gene Expression.

A luciferase reporter system was generated for testing microRNA-mediatedrepression in mammalian cells (FIG. 5 a). The firefly luciferase genewas fused to a mutated C. elegans lin-413′UTR (lin-413′UTR-delta) inwhich an 80-nt segment containing both let-7 complementary sites (green)was deleted, and replaced with a miR-223 perfect complementary site. Ina control reporter, the Renilla luciferase gene was fused to thelin-413′UTR-delta. All luciferase genes are under the control of theviral LTR promotor. Virus was produced by transfecting the constructsinto the BOSC23 viral packaging cell line and titered by infectingNIH3T3 cells. NIH3T3 cells stably expressing miR-223 or vector wereinfected with equal titer of fLuc-lin41UTRdelta+miR-223 target orfLuc-lin41UTRdelta−miR-223 target virus along with control virusrLuc-lin41UTRdelta. Four days after infection, luciferase activity wasmeasured and firefly luciferase activity was normalized using Renillareporter. The ectopically expressed miR-223 reduced by 4-fold expressionof a luciferase reporter gene that had a miR-223 complementary sitewithin its 3′ UTR, confirming that the miRNA was incorporated into amiRNA ribonucleoprotein complex (miRNP) and was capable of genesilencing (FIG. 5 b).

2. Expression of Artificial-microRNA/siRNAs Utilizing miRNA-ProcessingSignals

DNA vectors that express perfect complementary short hairpins RNAs arecommonly used to generate functional siRNAs. However, the efficacy ofgene silencing mediated by different short-hairpin derived siRNAs isinconsistent, and a substantial number of short-hairpin siRNA expressionvectors can trigger an anti-viral interferon response (Nature Genetics,2003, 34:263). Moreover, siRNA short-hairpins are typically processedsymmetrically, in that both the functional siRNA strand and itscomplement strand are incorporated into the RISC complex. Entry of bothstrands into the RISC can decrease the efficiency of the desiredregulation and increase the number of off-target mRNAs that areinfluenced. In comparison, endogenous miRNA processing and maturation isa fairly efficient process that is not expected to trigger an anti-viralinterferon response. This process involves sequential steps that arespecified by the information contained in miRNA hairpin and its flankingsequences (FIG. 2). Thus, we designed a novel strategy to expressartificial-microRNA/siRNAs utilizing miRNA-processing signals.

To this end, we selected the B1_C02-3 miRNA as a template forartificial-microRNA/siRNA expression (FIG. 6). Shown in FIG. 6 a are thepredicted stem-loop structure of B1_C02-3 miRNA and the actual sequenceof B1_C02-3 miRNA (Green letters). Based on cloning analysis, allB1_C02-3 clones are 23 nt in length, and no miR* strand was found incloning, while B1_C—O₂-3 was cloned over 18 times. Consistent withcloning analysis, northern analysis of ectopically expressed B1_C02-3revealed that the expressed miRNA was asymmetrically processed into adefined length (FIG. 6 b). In comparison, many miRNAs are processed intodifferent lengths with heterogeneity mostly seen in the 3′ end; a fewmiRNAs are also not asymmetrically processed in that both miR and miR*strands can be found at similar frequency. Thus, we hypothesized thatthe information for specific B1_C02-3 processing is defined by itsstem-loop and flanking sequences, and this information can be utilizedto express artificial-microRNA/SiRNA with defined length andasymmetrical strand accumulation.

Based on this hypothesis we designed the following strategy to expressartificial-microRNA/siRNAs utilizing B1_C02-3 templates (FIG. 7). Inbrief, a B1_C02-3 gene of about 273 nt in length containing the miRNAand 125 nt flanking sequences on each side of the miRNA was cloned intothe double-copy retroviral expression constructs. This construct caneffectively express B1_C02-3 (FIG. 6 b). A XhoI restriction enzyme sitewas introduced into the B1_C02-3 hairpin flanking sequence. This allowedus to easily replace the B1_C02-3 miRNA hairpin with anartificial-microRNA/siRNA hairpin. Some features of the B1_C02-3 miRNAhairpin structure, the loop and buldges (indicated with arrows in FIG.7) surrounding the miRNA stem, were preserved in allartificial-microRNA/siRNA hairpins. These features might be important todetermine the boundary of miRNA processing. A stem formed by anartificial-microRNA/siRNA sequence (with a length of 19 to 23 nt) andits perfect complement strand replaced the B1_C02-3 miRNA stem.Expression of the artificial-microRNA/siRNA and B1_C02-3 chimeric geneshould produce an asymmetric artificial-microRNA/SiRNA sequence with“CA” on its 5′end (FIG. 7). Because the expression and processing ofartificial-microRNA/SiRNA utilizes the miRNA processing machinery, thefinal artificial-microRNA/SiRNA products should be effectivelyincorporated into the functional miRNP/RISC.

To test this design, artificial-miRNAs/SiRNAs were designed to target21-nucleotide unique sequences in the firefly luciferase gene. Northernblot analyses (FIG. 8), using probes against sense and antisense strandsof the artificial-miRNAs/siRNAs, were used to determine the expressionand processing of SiLuc-1276 and SiLuc-311. In both cases, we observedasymmetrically processed SiLuc-1276 and SiLuc-311 products of 23 nt inlength. These results demonstrate that our design properly preservedprocessing information for B1_C02-3, and can be used to expressvirtually any desired artificial-miRNAs/siRNAs.

We analyzed the function of ectopically expressedartificial-miRNAs/siRNAs in their ability to mediate gene silencing. Aseries of artificial-miRNAs/SiRNAs expression constructs were designedto target a luciferase reporter gene. NIH3T3 cells stably expressingartificial-miRNAs/siRNAs or vector were infected with the fireflyLuciferase reporter along with a control Renilla reporter. Four daysafter infection, luciferase activity was measured and firefly luciferaseactivity was normalized using the Renilla reporter. The ectopicallyexpressed artificial-miRNAs/siRNAs against Luciferase can specificallyreduce firefly luciferase activity up to 90%. Four out of fiveconstructs could reduce reporter expression by at least 60%. Theseresults confirm that the expressed artificial-miRNAs/SiRNAs wasincorporated into a miRNA ribonucleoprotein complex (miRNP/RISC) and wascapable of gene silencing (FIG. 9).

3. Expression of miRNAs Using Different Promoters

Expression of microRNAs from Pol II and Pol III promoters. One strategyto express the miRNA is to include a sufficiently large DNA fragmentsuch that the miRNA is expressed under the control of its nativepromoter. A second strategy is to use heterologous promoters. We havedemonstrated that a microRNA can be effectively expressed and processedfrom a microRNA transcript containing the microRNA and correspondinggenomic flanking sequences using a pol III expression cassette. In FIG.10 we also show that pol II heterologous promoters can be used.

Inducible expression of microRNA using the TetOn system. (FIG. 10) Onestrategy to express the miRNA is to include a sufficiently large DNAfragment such that the miRNA is expressed under the control of itsnative promoter. A second strategy is to use heterologous promoters. Wehave demonstrated that a microRNA can be effectively expressed andprocessed from a microRNA transcript containing the microRNA andcorresponding genomic flanking sequences using a pol III expressioncassette. In FIG. 10 we also show that pol II heterologous promoters canbe used. Such promoters include those that are used in availablemammalian expression systems including tissue-specific promoters andinducible promoters. TetOn system is a commercial inducible expressionsystem from Clontech Inc. This is of particular interest because currentsiRNA expression systems utilize pol III promoters, which are difficultto adapt for inducible expression. In FIG. 10, we showed the expressionof miRNA (B1_C02-3) using the Clontech TetOn expression system. AB1_C02-3 gene with 500 nt flanking sequence was cloned into the pRev-TREvector. This construct was then packaged into retrovirus and used toinfect a Tet-On cell line expressing the reverse tetracycline-controlledtransactivator (rtTA). B1_C02-3 is inducibly expressed in response tovarying concentrations of the teratcycline derivate doxycycline (Dox).Similarly, this system can be used for expression of artificialmicroRNAs.

4. miRNAs Modulate Hematopoietic Lineage Differentiation

Expression of microRNAs in hematopoietic tissues. To investigate whethermicroRNAs play a role in mammalian development and in particular mightregulate mammalian hematopoiesis, we cloned more than 70 uniquemicroRNAs from mouse bone marrow. miR-132s, the microRNA found at abreakpoint of a t(8:17) translocation associated with an aggressiveB-cell leukemia (Gauwerky et al., 1989, Proc. Natl. Acad. Sci. USA,86:8867-8871), was expressed in all four hematopoietic tissues tested:fetal liver, and adult bone marrow, spleen, and thymus, with little orno expression in the non-hematopoietic tissues. The lung was the onlyother tissue with appreciable miR-132s expression (FIG. 11). Expressionin E13 fetal liver suggested that miR-132s might function in earlyhematopoietic development. Expression of mature miR-132s was highest inthymus, the primary lymphoid organ that mainly contains T-lymphocytes.Expression was much lower in bone marrow, which consists ofhematopoietic stem cells and myeloid, erythroid and lymphoid cells at avariety of differentiation stages, and spleen. Interestingly,accumulation of the presumed miR-132 precursor (˜60 nt band, FIG. 11)was high and the ratio of mature to precursor 21 nt RNAs varied indifferent tissues, suggesting post transcriptional regulation ofmicroRNA expression at the level of precursor processing or RNAstability.

miR-223 was very strongly expressed in bone marrow and was detectable inspleen but essentially absent in E13 fetal liver, thymus, and all otheradult mouse tissues tested (FIG. 11). miR-181 was very stronglyexpressed in thymus and was detectable in bone marrow and spleen butessentially negative in E13 fetal liver and all other mouse tissuestested except for high expression in brain and lung. miR-181 and miR-223expression in fetal liver was barely detectable, suggesting that theyonly function in adult hematopoiesis. Thus miR-181, miR-223 and miR-132sare differentially expressed in hematopoietic tissues and theirexpression is regulated during development.

microRNAs are Regulated in Hematopoietic Lineage Commitment.

Because bone marrow, spleen, and thymus, each have specialized functionsin adult hematopoiesis and comprise significantly different cell types,the differential expression of the miRNAs in these complex tissuessuggested that individual hematopoietic cell types might differentiallyexpress the miRNAs. Indeed, when cells within bone marrow were sortedbased on lineage markers, they were found to differentially express thehematopoietic miRNAs (FIG. 12). In contrast, expression of miR-16, anmiRNA seen in a broad range of tissues, was more constant.

Mature miR-181 expression in the bone marrow cells was detectable inundifferentiated progenitor cells (Lin⁻) and up-regulated in thedifferentiated B-lymphocytes, marked by the B220 surface antigen. Inother differentiated lineages, miR-181 expression did not increase overthat seen in Lin⁻ cells. Note that sorted lineage cell populations areabout 85% pure, thus some residual miRNA signal in the other lineagesmight be caused by contamination of B220⁺ cells. Expression of themiR-181 precursor was at similar levels in all lineages, suggesting thatthe differential accumulation of the mature miR-181 during hematopoieticlineage commitment might be regulated at the level of miRNA processingor the rate of turnover.

miR-223 expression was confined to myeloid lineages (Gr-1⁺ and Mac-1⁺)with barely detectable expression in T- and B-lymphoid and erythroidlineages (CD3e⁺, B220⁺, and Ter119⁺, respectively; FIG. 12). Thisobservation is consistent with miR-223 expression in bone marrow but notspleen and thymus (FIG. 11). miR-132s expression was lowest in theerythroid lineage (Ter-119⁺) and highest in myeloid lineages (Gr-1⁺ andMac-1⁺), consistent with its ubiquitous expression in bone, spleen andthymus (FIGS. 11 and 12).

For each of the miRNAs, specific expression was validated by thereduction of correspondent miRNA expression in the reciprocallineage-depleted cell populations (FIG. 12, right panels). In addition,expression of all four miRNAs was low in Lin⁻ cells relative to theirpreferred Lin⁺ cell populations, suggesting that these miRNAs wereinduced upon lineage commitment and differentiation.

microRNAs are Capable of Modulating Hematopoietic Lineage Commitment.

Hematopoietic progenitor cells from mouse bone marrow were infected withthe viral vectors expressing either miR-181, miR-223, miR-132s, or acontrol miRNA, miR-30. Interestingly, ectopic expression of each ofthese microRNAs in hematopoietic bone marrow Lin⁻ progenitor cells havedifferential effects on the differentiation of bone marrow progenitorcells, particularly the differentiation of T- or B-lymphoid lineages,which are indicated by the expression of Thy-1.2 or CD-19 cell surfaceantigens (FIG. 13).

About 23±2.5% (n=12) or 12±2.3% (n=12) differentiated cells expressedThy-1.2 or CD-19 antigen, respectively, when infected with controlvector. Over-expression of miR-30 did not significantly alter the ratioof T- and B-lymphoid lineage cells, in that the marker expression wasessentially unchanged compared to that of cells infected with the emptyvector. This indicated that merely expressing an arbitrary microRNA didnot markedly influence lymphoid differentiation. In contrast, about21±4.2% (n=12) or 26±1.7% (n=12) differentiated cells expressed Thy-1.2or CD-19 antigen, respectively, when infected with miR-181 expressionvirus. Thus, expression of miR-181 substantially affected the lineagedifferentiation, resulting in more than a 2-fold increase in B-lymphoidlineage with little change in T-lymphoid lineage. In contrast, ectopicexpression of miR-132s and miR-223 resulted in opposite effects, with a30˜40% increase in the T-lymphoid lineage and a slight reduction in theB-lymphoid lineage, and more significant change in the ratio ofT/B-lineage cells which increased from ˜2-fold (vector) to 2.5 (miR-223)or 4.5-fold (miR-132s), respectively.

Modest effects were seen when analyzing these cells for myeloid lineagemarkers (FIGS. 13 c and d). Neutrophils are Mac-1 and Gr-1double-positive cells (Mac-1⁺ Gr-1⁺), whereas monocytes are Mac-1positive and Gr-1 negative-to-low (Mac-1⁺ Gr-1^(−/low)). Non-myeloidcells were Mac-1 and Gr-1 double-negative (Mac-1⁻ Gr-1⁻); they mostlyexpressed Thy-1.2 or CD-19 lymphoid markers. Overexpression of thecontrol miR-30 had little if any effect on Mac-1 and Gr-1 expression.Over-expression of either miR-132s or miR-181 led to a small decrease inMac-1⁺ Gr-1^(−/low) cells but expression of miR-181 led to a noticeableincrease in Mac-1⁺ Gr-1⁺ cells.

The demonstration that certain miRNAs are differentially expressed inhematopoietic lineages in vivo and are able to alter lineage commitmentin vitro provides solid evidence that microRNAs represent a class ofmolecules that regulate mammalian development. This supports the notionthat the roles of translational regulation in hematopoiesis and, morebroadly, vertebrate development might have been under-appreciated.Furthermore, modulating the expression of miRNAs can have therapeuticutility.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. Variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.The advantages and objects of the invention are not necessarilyencompassed by each embodiment of the invention.

1. A precursor microRNA molecule, comprising: an isolated nucleic acidcomprising: a stem-loop structure, wherein a microRNA sequence isincorporated into a stem of the stem-loop structure, and a microRNAflanking sequence flanking at least one end of the stem-loop structure.2. The precursor microRNA molecule of claim 1, wherein the microRNAflanking sequence is between 40 and 2,000 nucleotides in length.
 3. Theprecursor microRNA molecule of claim 1, wherein the microRNA sequenceand the microRNA flanking sequence are derived from the same microRNAgene.
 4. The precursor microRNA molecule of claim 1, wherein themicroRNA sequence and the microRNA flanking sequence are not derivedfrom the same microRNA gene.
 5. The precursor microRNA molecule of claim4, wherein the microRNA sequence is an artificial microRNA sequence. 6.The precursor microRNA molecule of claim 1, wherein the precursormicroRNA molecule includes at least two stem-loop structures.
 7. Theprecursor microRNA molecule of claim 1, wherein the microRNA flankingsequence is between 40 and 4,000 nucleotides in length.
 8. The precursormicroRNA molecule of claim 1 wherein the molecule has the followingnucleic acid sequence

wherein X₁ and X₂ are nucleotides and wherein N₁ and N₂ are nucleicacids of 16-28 nucleotides in length and wherein N₁ and N₂ have at leastpartial complementarity.
 9. The precursor microRNA molecule of claim 8,wherein X₁ and X₂ are each between 40 and 4,000 nucleotides in length.10. The precursor microRNA molecule of claim 1, wherein the precursormicroRNA molecule has microRNA flanking sequences flanking each end ofthe stem-loop structure.
 11. A precursor microRNA molecule, comprising:a nucleic acid comprising: a stem-loop structure, wherein a microRNAsequence is incorporated into a stem of the stem-loop structure, and, amicroRNA flanking sequence flanking at least one end of the stem-loopstructure, wherein the microRNA sequence and the microRNA flankingsequence are not derived from the same microRNA gene.
 12. The precursormicroRNA molecule of claim 11, wherein the microRNA flanking sequence isbetween 40 and 2,000 nucleotides in length.
 13. The precursor microRNAmolecule of claim 11, wherein the microRNA flanking sequence is between40 and 4,000 nucleotides in length.
 13. A method of altering theproductive utilization of a target mRNA, comprising: contacting a cellwith a vector capable of expressing a precursor microRNA of any one ofclaims 1-8 or 11-13 wherein the precursor microRNA includes a microRNAsequence capable of altering the productive utilization of the targetmRNA.
 15. The method of claim 13, wherein the precursor microRNA isspecific for a cancer-associated RNA.
 16. The method of claim 13,wherein the precursor microRNA is specific for a viral RNA.
 17. Themethod of claim 13, wherein the method is performed in vivo.
 18. Themethod of claim 17, wherein the cell is in a subject having cancer. 19.The method of claim 17, wherein the cell is in a subject having aninfection.
 20. A method of altering the productive utilization of atarget mRNA, comprising: contacting a cell with a vector capable ofexpressing a mature microRNA that is not naturally expressed in thecell, wherein the mature microRNA is expressed at a level sufficient tocause at least a 10 fold reduction in accumulation of a protein from thetarget mRNA.
 21. A method of altering the productive utilization of atarget mRNA in primary cells, comprising: contacting a primary cell witha vector capable of expressing a mature microRNA that is not naturallyexpressed in the cell, wherein the mature microRNA is expressed at alevel sufficient to cause a reduction in accumulation of a protein fromthe target mRNA in the primary cell.
 22. The method of claim 21 whereinthe primary cell is in vivo.
 23. A composition comprising a vector forproducing a precursor microRNA wherein the vector includes a sequenceencoding a precursor microRNA, including a microRNA sequence andmicroRNA flanking sequences, and at least one promoter element.
 24. Thecomposition of claim 23, wherein the vector is a viral vector.
 25. Thecomposition of claim 24, wherein the viral vector is a retroviralvector.
 26. The composition of claim 23, wherein the at least one of thepromoter is an inducible promoter.
 27. The composition of claim 23,wherein the precursor microRNA molecule is selected from the group ofprecursor microRNA molecules of claim 1 and
 11. 28. The composition ofclaim 23, wherein the vector has nucleic acid sequence of SEQ ID NO. 1or variants thereof.
 29. The composition of claim 28, wherein the vectorhas nucleic acid sequence of SEQ ID NO.
 1. 30. A host cell transfectedwith the vector of claim
 23. 31. A method for detecting precursormicroRNA expression, comprising: incorporating a precursor microRNA intoa reporter system, transfecting a host cell with the reporter system,and detecting expression of a reporter gene product to detect theexpression of precursor microRNA by its effect on translationalrepression.
 32. The method of claim 31, wherein the reporter systemincludes a firefly luciferase reporter gene.
 33. A method for modulatinghematopoiesis, comprising: contacting a hematopoietic cell with a vectorcapable of expressing a precursor microRNA of any one of claims 1-8 or11-13 wherein the precursor microRNA includes a microRNA sequencecapable of altering accumulation of a protein involved in hematopoiesis.